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Tandem intermolecular [4 + 2] cycloadditions are catalysed by glycosylated enzymes for natural product biosynthesis

Nature Chemistry volume 15, pages 1083–1090 (2023)Cite this article

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Abstract

Tandem Diels–Alder reactions are frequently used in the construction of polycyclic ring systems in complex organic compounds. Unlike the many Diels−Alderases (DAases) that catalyse a single cycloaddition, enzymes for multiple Diels–Alder reactions are rare. Here we demonstrate that two calcium-ion-dependent glycosylated enzymes, EupfF and PycR1, independently catalyse sequential, intermolecular Diels–Alder reactions in the biosynthesis of bistropolone-sesquiterpenes. We elucidate the origins of catalysis and stereoselectivity within these DAases through analysis of enzyme co-crystal structures, together with computational and mutational studies. These enzymes are secreted as glycoproteins with diverse N-glycans. The N-glycan at N211 in PycR1 significantly increases the affinity to the calcium ion, which in turn regulates the active cavity, making it specifically interact with substrates to accelerate the tandem [4 + 2] cycloaddition. The synergistic effect of the calcium ion and N-glycan on the catalytic centre of enzymes involved in secondary metabolism, especially for complex tandem reactions, can extend our understanding of protein evolution and improve the artificial design of biocatalysts.

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Fig. 1: EupfF and PycR1 are TDA enzymes.
Fig. 2: Crystal structures and relative activity of EupfF and PycR1.
Fig. 3: DFT calculations, MD simulations and proposed catalytic cycles.

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Data availability

All the data that support the findings of this study are available within the paper and its supplementary information. The atomic coordinates of EupfF, PycR1, PycR1 with 4′ and PycR1 with 2 and 7 have been deposited in the Protein Data Bank (http://www.rcsb.org) under accession codes 7X36, 7X2N, 7X2X and 7X2S, respectively. Source data are provided with this paper.

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Acknowledgements

This work was supported financially by the National Science Fund for Distinguished Young Scholars (no. 82225042 to Y.H.), the CAMS Innovation Fund for Medical Sciences (CIFMS, no. 2021-I2M-1-029 to Y.H.), the National Key Research and Development Program of China (no. 2018YFA0901900 to J.Z.), the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (no. 2022-RC350-05 to Y.H.), and the National Institutes of Health (GM 124480 to K.N.H.). All calculations were performed on the Hoffman2 cluster at UCLA and the UCLA Institute of Digital Research and Education (IDRE). We thank the staff members at beamlines BL18U1 and BL19U1 of the Shanghai Synchrotron Radiation Facility for help with data collection, and we thank H. Liu and Y. L. Zhang for their assistance with this project. We also thank H. L. Li and S. J. Xia from Sun Yat-sen University for carrying out the native MS analysis.

Author information

Author notes

  1. These authors contributed equally: Jiawang Liu, Jiayan Lu, Chen Zhang, Qingyang Zhou.

Authors and Affiliations

  1. State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, People’s Republic of China

    Jiawang Liu, Chen Zhang, Changhui Shang & Youcai Hu

  2. State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, People’s Republic of China

    Jiayan Lu

  3. CAS Key Laboratory of Quantitative Engineering Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, People’s Republic of China

    Jiayan Lu & Jiahai Zhou

  4. Department of Chemistry and Biochemistry, University of California, Los Angeles, CA, USA

    Qingyang Zhou, Cooper S. Jamieson & K. N. Houk

  5. NHC Key Laboratory of Biosynthesis of Natural Products, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, People’s Republic of China

    Youcai Hu

  6. CAMS Key Laboratory of Enzyme and Catalysis of Natural Drugs, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, People’s Republic of China

    Youcai Hu

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Contributions

J.L., K.N.H., J.Z. and Y.H. conceived the idea and designed the experiments for the study. J.L. performed in vivo and in vitro assays, mutation experiments, as well as compound isolation and characterization. J.-Y.L. preformed structural biology. C.Z. cloned and purified the proteins and constructed the mutated and expressed plasmid. Q.Z. and C.S.J. performed computational experiments. C.S. performed partial compound isolation. J.L., J.-Y.L., C.Z., Q.Z., K.N.H., J.Z. and Y.H. analysed and prepared the manuscript.

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Correspondence to K. N. Houk, Jiahai Zhou or Youcai Hu.

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Extended data

Extended Data Fig. 1 Feeding experiments.

Substrates were fed to A. nidulans (AN.) cultures expressing eupf genes; fed on day 2 (left column) or on day 4 (right column). (i) stipitaldeyhde 5 was fed to A. nidulans co-expressing eupfDEFG. Compounds 4 and 5 were co-fed to A. nidulans co-expressing (ii) eupfEF or (iii) eupfE or (iv) eupfF, or (v) control. (vi) 4 was fed to A. nidulans co-expressing eupfABCEF (without eupfDG). (vii) 5 and 2 were co-fed to A. nidulans co-expressing eupfEF. (viii) 2 was fed to A. nidulans co-expressing eupfABCEF. Liquid chromatography analysis was shown at characteristic ultraviolet (UV) absorption of 360 nm or 280 nm. Control: strains with empty vector. A ring-contraction product pughiinin A (8) appeared when compounds 4-5 were fed to A. nidulans at day 2. Compound 8 may be derived from 2 by A. nidulans30.

Extended Data Fig. 2 Overall structure of PycR1 (light blue, left) and EupfF (orange, right).

(a) PycR1 has two N-glycans: N43 and N211. (b) EupfF has four N-glycans: N45, N68, N213, and N300. N-glycans are shown as colored light blue in PycR1, and sticks, colored orange in EupfF. Both PycR1 and EupfF have one Ca2+, colored violet. Enlarged view of the Ca2+ binding site in PycR1 (c) and EupfF (d). Coordinate bond interactions are indicated with violet dashed lines. The Ca2+ is bound by coordinate bond interactions with E48, Q158, N228, and D275 in PycR1. For EupfF, the Ca2+ is bound by coordinate bond interactions with E50, Q156, N225, and D273.

Extended Data Fig. 3 Mutation of Ca2+ binding residues in PycR1.

The PycR1 residues E48, Q158, N228, and D275 were selected for single or double mutation. Enzymatic reactions were performed with 10 μM enzyme (WT, or variants) and incubated with 100 μM 6, along with 10 μM 4 and 10 μM 4’ (left column) or 10 μM 2’ (right column) in PBS buffer for 90 mins. The experiments were repeated three times independently, and similar results were obtained each time.

Extended Data Fig. 4 Structural alignment between PycR1 (light blue) and EupfF (orange).

(a) The root mean squared deviation (r.m.s.d) for Cα atoms is 0.365 Å; (b) Enlarged view of the glycosylation site N211 in PycR1 and N213 in EupfF.

Extended Data Fig. 5 MD simulations and ONIOM calculations of the glycan-free and glycosylated PycR1 and corresponding dehydration TS.

(a) MD simulations of glycan-free and glycosylated PycR1. (b) Coordination bond interactions of Ca2+ with E48, Q158, N228, and D275 in PycR1. For glycan-free enzyme, the bond between Ca2+ and Q158 is diminished. (c) Dehydration TS of 6 in glycan-free PycR1 (left column) and glycosylated PycR1(right column) calculated by ONIOM. In glycan-free enzyme, the dehydration TS has a higher barrier.

Extended Data Fig. 6 Computed transition state (TS) energies for the pycnidione series (a) and the eupenifeldin series (b).

(a) The transition state (TS) energies to form experimental observed epolone B (2’) and pycnidione (1’). The competitive transition state pyc-TS2 for diastereoisomeric monoadduct 2’-a, and pyc-TS4,5,6 for bis-adducts 1’-a/b/c. The reaction barriers between the transition state pyc-TS1 and pyc-TS2 was 0.8 kcal· mol−1, while the reaction barriers between the transition state pyc-TS3 and pyc-TS4 was 6.6 kcal· mol−1; (b) The transition state eup-TS1 and eup-TS2 for monoadducts 2 (experimental product) and 2-a, and eup-TS3-TS6 for bis-adducts 1 (experimental product) and 1-a/b/c. The reaction barrier for the transition state eup-TS1 was 1.9 kcal· mol−1 lower than eup-TS2, and the reaction barriers for the transition state eup-TS3 was 1.3 kcal· mol−1 lower than eup-TS4. The energies shown in the figure are free energies, and were calculated using ωB97X-D/def2-QZVPP//ωB97X-D/def2-SVP.

Extended Data Fig. 7 MD simulations of the computed transition state in PycR1.

(a) MD simulations of pyc-TS1 that lead to monoadduct 2’ in PycR1 with Ca2+. Diene 7 interacts with W46, S63, and R94, while dienophile 4’ undergoes a weak interaction with Y223; (b) MD simulations of pyc-TS1 in PycR1 without Ca2+. Upon removal of Ca2+, there is only a weak hydrogen bond between 7 and S63; c) MD simulations of pyc-TS3 in PycR1 without Ca2+. In this case, 7 does not interact with S63 or R94, while 2’ only interacts with N293; d) MD simulations of pyc-TS4 in PycR1 with Ca2+. No significant interaction between pyc-TS4 and nearby residues was predicted. PycR1 is shown as a cartoon (colored gray); 7 (colored cyan), 4’ (colored wheat), and 2’ (colored yellow) are shown as sticks.

Extended Data Fig. 8 Mutation of key residues in active cavity of PycR1 and EupfF.

The residues W46, S63, R94, Y176, S242, N293, and N341 in PycR1, and the corresponding EupfF residues W48, S65, R96, Y174, Q239, N291, and N340 were selected for mutation. Enzymatic reactions were performed with 10 μM enzyme (WT, or variants) and incubated with 100 μM 6, and (a) 10 μM 4 and 10 μM 4’ or (b) 10 μM 2 or 10 μM 2’ in buffer for 90 mins. The experiments were repeated three times independently, and similar results were obtained each time.

Extended Data Fig. 9 MD simulation of mutation of key residues in active cavity of PycR1.

(a) MD simulations of the Y176A mutant with pyc-TS1. It shows a representative structure of pyc-TS1 in Y176A. The results indicate that removal of the phenyl group destabilizes the hydrogen bond between Oxygen 2 in 7 and R94. (b) MD simulation of pyc-TS3 with the N341K mutant. The structure was obtained upon docking and after minimization and equilibrium processing. (c) MD simulations of the S242M mutant with pyc-TS3. It shows the representative structure of pyc-TS3 in S242M PycR1. Introduction of the bulky thiomethyl group change the loop near V225, making V225 far apart from Oxygen 4 in 2’, and breaking this hydrogen bond. (d) MD simulations of the S242Q mutant with pyc-TS1 (left) and eup-TS1 (right). Diene 7 is omitted for clarity. The left is an overlay of the representative structures of S242Q-4’ (green) and WT-4’ (cyan). The right presents structures of S242Q-4 (green) and WT-4’ (cyan). The 4’ in pyc-TS1 appears to adopt a highly distorted conformation, suggesting substantial destabilization of pyc-TS1. The calculated distortion energies also show that this conformation is unfavored compared to eup-TS1 (by about 6.4 kcal/mol). The distortion energy for S242Q-4’ is defined as the electronic energy difference between the representative conformation of 4’ in simulation and pyc-TS1, which represents the energy required to distort the 4’ into geometry in enzyme environment.

Extended Data Fig. 10 Multiple sequence alignment of hDAases (a) and engineering modification (b).

(a) Red triangle, blue heptagon shape, green column shape, purple incomplete round shape, and pink hourglass represent the glycosylated residues, the tropolone binding residues, the Ca2+ binding residues, the monoadduct binding residues, the unique residues in AsR5 (a putative DAase in the biosynthesis of Xenovulene A31), respectively. (b) The mutation in EupfF and PycR1 can achieve to control the selection of substrate for 4’/4 and the number of reaction steps.

Supplementary Information

Supplementary Information

Complementary experimental procedures 1–12, Supplementary Tables 1–19 and Figs. 1–73, computational analysis, references.

Reporting Summary

Supplementary Data 1

Source data for graphs in Supplementary Fig. 10.

Supplementary Data 2

Source data for graphs in Supplementary Fig. 13.

Supplementary Data 3

Source data for graphs in Supplementary Fig. 15d.

Supplementary Data 4

Source data for graphs in Supplementary Fig. 18.

Supplementary Data 5

Source data for graphs in Supplementary Fig. 19.

Supplementary Data 6

Source data for graphs in Supplementary Fig. 21.

Supplementary Table

Source data for Supplementary Table 2.

Source data

Source Data Fig. 2

Source data for graph in Fig. 2.

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Liu, J., Lu, J., Zhang, C. et al. Tandem intermolecular [4 + 2] cycloadditions are catalysed by glycosylated enzymes for natural product biosynthesis. Nat. Chem. 15, 1083–1090 (2023). https://doi.org/10.1038/s41557-023-01260-8

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